A cell sorting technique based on flow cytometry has been known as a method for selecting specific individual cells or other fine particles from a group of cells or the like [e.g., Nakauchi, Hiromitsu (University of Tsukuba, medical immunology), supervision, “Flow Cytometry Jiyu Jizai (flexible flow cytometry)”, a separate volume of “Cell Technology”, Shujun-Sha, Jul. 1, 1999, pp. 3-23].
In this technique, a suspension of target cells combined with an antibody that has been previously labeled with a fluorescent dye or like substance is made to flow, and first, the target cells in the flow path are irradiated with an excitation light selected in accordance with the fluorescent dye used for labeling, and the wavelength and intensity of the fluorescence or scattered light emitted from each cell are analyzed to distinguish the target cells. Subsequently, a voltage is applied to the distinguished cells with specific properties to electrically charge the cells, which are then subjected to discrimination, quantification, static analysis, etc., by using deflecting electrodes.
Since the technique is capable of processing a large amount of cells at a high speed in the living state, it is widely applied in the fields of immunology, hematology, genetic engineering, etc., for harvesting and isolating various cultured cells, cloning and proliferating specific cells, fractioning cells that express a specific antigen on their surface, conducting a kinetic analysis of cell membrane molecules, analyzing chromosomes, and other purposes. In particular, the technique is becoming indispensable for analyzing cell kinetics. Besides, this technique is beginning to be used in the clinical field, for example, to analyze solid components of urine.
The cell analysis and separation device used in the flow cytometry cell sorting technique is called a cell sorter (fluorescence-activated cell sorter, FACS). This device has an analysis portion that performs the analysis of fluorescence or other light mentioned above, and a sorting portion provided downstream of the analysis portion. Typically, a droplet charging system is employed as the sorting portion.
The cell (particle) sorting by the droplet charging system is based on, for example, the following principle. Cells in droplets in which scattered light and fluorescence are detected upon irradiation with a laser beam are positively or negatively charged, immediately before the liquid flow containing the cells is divided into droplets. When the charged droplets containing cells are allowed to fall so as to pass between two polarizing electrode plates that are different in potential, the droplets are drawn to the polarizing plates and deflected. Since the droplets containing uncharged cells and drops containing cells other than the desired cells fall perpendicularly, droplets containing only the desired cells can be separated and recovered.
However, the technique using such a cell sorter requires an ultrasonic generator to produce droplets each containing one cell (particle), and thus has disadvantages in that the cell sorter is not only expensive but also requires complicated operation and maintenance procedures and is incapable of sorting various kinds of fine particles at a time. Specifically, when using electrode plates, the cells can be electrically distinguished only by charging either positively or negatively, and therefore only two types of cells can be distinguished. Further, since the flow cell and nozzle are used repeatedly, impurities may be mixed in. Furthermore, problems such as atmospheric contamination by hazardous substances may occur when ultrasonically atomizing the cell suspension during the cell sorting.
In order to solve the problems of cell sorters, such as the extremely high price, overly complicated operation and maintenance procedures, and admixture of impurities, a method has been proposed which can be performed at low cost without the admixture of impurities. In this method, a microfabridated channel is formed on a substrate made of glass or a polymeric material, and cells or like fine particles are placed in a liquid flow and subjected to flow cytometry in the channel, thereby sorting the desired fine particles (a technique using a microchip) (e.g., Anne. Y. Fu et al., “A microfabricated fluorescence-activated cell sorter”, Nature Biotechnology, Vol. 17, November 1999, pp. 1109-1111; and Anne Y. Fu et al., “An Integrated Microfablicated Cell Sorter”, Analytical Chemistry, Vol. 74, No. 11, Jun. 1, 2002, pp. 2451-2457).
In this cell sorting operation using a microchip, a T-shaped channel is formed, and the desired cells are sorted from other cells by switching the flow of the solution that carries the cells (flow path selection control).
However, in the proposed method, the flow of the liquid can be switched only once, and thus only one kind of fine particles can be separated. One idea is that switching the flow direction two or more times can separate multiple kinds of fine particles, but since the response time of switching the liquid flow is too long, two or more flow switching steps require two or more liquid feed pumps. The connection of the pumps to the chip and the opening and closing of the valves are too complicated to put the idea into practice.